Tarsal morphology and attachment ability of the codling moth Cydia pomonella L. (Lepidoptera, Tortricidae) to smooth surfaces

Despite several studies on the attachment ability of different insect taxa, little is known about this phenomenon in adult Lepidoptera. In this study we combined morphological and experimental analyses of tarsal adhesive devices and the attachment ability of the codling moth Cydia pomonella (L.) (Lepidoptera, Tortricidae) to smooth surfaces. Pretarsi of C. pomonella attach to smooth substrates by means of their smooth, flexible and well developed arolia. Using the centrifugal force measurement technique, friction forces of males and females were assessed on hydrophobic and hydrophilic glass surfaces. Adults of both sexes generated similar forces in spite of the noticeable difference in their body masses. That is why males showed significantly higher safety factors (attachment force divided by body weight) compared to those of females. Hydrophobicity of the substrate had no considerable effect on friction forces. For females, friction forces (sliding parallel to the substrate plane) were compared with adhesive forces (pulling off perpendicularly from the substrate plane) measured on Plexiglas surfaces. It can be concluded that the attachment system of C. pomonella is rather robust against physico-chemical properties of the substrate and is able to achieve a very good attachment on vertical and horizontal substrata.     

Changes in tarsal morphology and attachment ability to rough surfaces during ontogenesis in the beetle Gastrophysa viridula (Coleoptera,Chrysomelidae)

Insects live in a three-dimensional space, and need to be able to attach to different types of surfaces in a variety of environmental and behavioral contexts. Adult leaf beetles possess great attachment ability due to their hairy attachment pads. In contrast, their larvae depend on smooth pads to attach to the same host plant. We tested friction forces generated by larvae and adults of dock leaf beetles Gastrophysa viridula on different rough surfaces, and found that adults generate much higher attachment to various substrates than larvae, but are more susceptible to completely losing attachment ability on surfaces with “critical” roughness. Furthermore, sex-specific setal morphology has the effect that attachment forces of male adults are generally higher than those of females when adjusted for body weight. The results are discussed in the context of development, ecology, and changing behavioral strategies of successive life stages.     

Adhesive and frictional properties of tarsal attachment pads in two species of stick insects (Phasmatodea) with smooth and nubby euplantulae

In the present study, the tarsal attachment pads (euplantulae) of two stick insect species (Phasmatodea) were compared. While the euplantulae of Cuniculina impigra (syn. Medauroidea extradentata) are smooth, those of Carausius morosus bear small nubs on their surfaces. In order to characterize the adhesive and frictional properties of both types of euplantulae, adhesion and friction measurements on smooth (Ra=0.054 μm) and rough (Ra=1.399 μm) substrates were carried out. The smooth pads of C. impigra generated stronger adhesion on the smooth substrate than on the rough one. The adhesive forces of the structured pads of C. morosus did not differ between the two substrates. Friction experiments showed anisotropy for both species with higher values for proximal pulls than for distal pushes. In C. impigra, friction was stronger on the smooth than on the rough surface for both directions, whereas in C. morosus friction was stronger on the smooth surface only for pushes. This shows that smooth attachment pads are able to generate relatively stronger adhesion and friction on a flat smooth surface than on a rough one. In contrast, nubby pads have similar adhesion on both substrates, and also show no difference in friction in the pulling direction. This leads to the conclusion that smooth pads are specialized for rather smooth substrates, whereas nubby pads are better adapted to generate stronger forces on a broader range of surfaces.     

Effects of surface topography and chemistry of Rumex obtusifolius leaves on the attachment of the beetle Gastrophysa viridula

Broad-leaved dock, Rumex obtusifolius L. (Polygonaceae), is the main host plant of the green dock beetle, Gastrophysa viridula Degeer (Coleoptera: Chrysomelidae). Adult beetles are able to attach and walk on leaves of this plant. Leaf surface is rather uneven, because of irregularly shaped prominent epidermal cells with a maximum height of about 9 µm. The surface is covered with a smooth epicuticular wax layer having relatively low free surface energy (FSE). The aim of this study was to measure beetle attachment force applying a 'centrifugal technique' on adult insects on the leaf surface and other substrates, in order to understand the effect of surface architecture and its physicochemical properties on insect attachment. We compared forces on an adaxial leaf surface with forces on a smooth silanized glass plate having low FSE, and on a polishing paper having slightly lower FSE and similar surface roughness (asperities' size = 9 µm). Smooth plate made of normal untreated clean glass with high FSE was used as a reference substrate. On the leaf surface and the polishing paper, attachment forces were lower compared to both glass samples. There was no significant difference between force values on leaves and the polishing paper, but on both glass surfaces they were significantly higher than on the other substrates. Hydrophobicity alone explains a decrease in attachment force of the beetle, but when combined with roughness the decrease in force is four times greater.     

Impact of chemical manipulation of tarsal liquids on attachment in the Colorado potato beetle, Leptinotarsa decemlineata

Insect tarsal attachment forces are thought to be influenced by the viscosity and surface tension of a thin film of adhesive liquid (wet adhesion). In beetles, this fluid has been shown to be composed mainly of lipophilic substances that are similar to the cuticular lipids. In this study we investigate whether and how the chemical composition of footprint lipids affects attachment forces in the Colorado potato beetle, Leptinotarsa decemlineata. After application of standardised mixtures of synthetic n-alkanes or alkenes, or a concentrated hydrocarbon extract to the surface of the elytra, we tested the beetles’ attachment performance using a beam force transducer. The results show that only the unsaturated components, but not the straight-chained alkanes reduced friction forces, confirming that attachment performance is influenced by the chemical composition of the adhesive secretion. We estimated the volume of footprint droplets and calculated a mean thickness of the liquid layer of 0.04 μm. The measured friction exceeded the viscous and capillary force expected for a film of this thickness. Therefore, alternative mechanisms (i.e. shear-thinning and solid-like behaviour) for the generation of attachment forces and their dependence on the chemical composition of the liquid are discussed.     

Sexual dimorphism in the attachment ability of the Colorado potato beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) to rough substrates

Many representatives of the beetle family Chrysomelidae exhibit a distinctive sexual dimorphism in the structure of adhesive tarsal setae. The present study demonstrates the influence of surface roughness on the friction force of Leptinotarsa decemlineata males and females. The maximum friction force of individual beetles was measured on epoxy resin surfaces (smooth and with asperities ranging from 0.3 to 12.0 microm) using a centrifugal force tester. On the smooth surface, no considerable differences between males and females were found, whereas on rough surfaces, females attached significantly (up to two times) stronger than males. Clawless beetles generated lower forces than intact ones, but demonstrated similar differences between males and females. The results indicate that the female adhesive system has its main functional trait in a stronger specialisation to rough plant surfaces whereas the adhesive system of males possess a certain trade-off between attachment to rough plant surfaces during locomotion on vegetation and to the smooth surface of the female elytra, while mating.     

The effect of surface roughness on claw and adhesive hair performance in the dock beetle Gastrophysa viridula

Abstract Natural adhesive systems are adapted to attach to rough surfaces, but the underlying mechanisms have not been fully clarified. Attachment forces for the beetle Gastrophysa viridula were recorded on epoxy casts of surfaces with different roughness using a centrifuge device. Replicas were made of standardized polishing paper with asperity sizes ranging from 0.05 to 30 μm and of dock leaves (Rumex obtusifolius). Beetles adhered with a safety factor of up to 36 times body weight on smooth substrates or on casts of leaves of their host plant. On the rough substrates, forces were much lower and a minimum at small scale roughness (0.05 μm asperity size, with a mean safety factor of 5) was observed. Removal of the claws led to a significant reduction in force for rough substrates with asperity sizes ≥ 12 μm. Attachment forces of the hairy adhesive system itself (without the claws) slightly increased from small‐scale to large‐scale surface roughness, but remained below the level seen on the smooth substrate. This is explained by the inability of setal tips to make full contact to the surface.     

The viscoelasticity of membrane tethers and its importance for cell adhesion

Cell adhesion mechanically couples cells to surfaces. The durability of individual bonds between the adhesive receptors and their ligands in the presence of forces determines the cellular adhesion strength. For adhesive receptors such as integrins, it is a common paradigm that the cell regulates its adhesion strength by altering the affinity state of the receptors. However, the probability distribution of rupture forces is dependent not only on the affinity of individual receptor-ligand bonds but also on the mechanical compliance of the cellular anchorage of the receptor. Hence, by altering the anchorage, the cell can regulate its adhesion strength without changing the affinity of the receptor. Here, we analyze the anchorage of the integrin VLA-4 with its ligand VCAM-1. For this purpose, we develop a model based on the Kelvin body, which allows one to quantify the mechanical properties of the adhesive receptor's anchorage using atomic force microscopy on living cells. As we demonstrate, the measured force curves give valuable insight into the mechanics of the cellular anchorage of the receptor, which is described by the tether stiffness, the membrane rigidity, and the membrane viscosity. The measurements relate to a tether stiffness of k_t = 1.6 μN/m, an initial membrane rigidity of k_i = 260 μN/m, and a viscosity of μ = 5.9 μN·s/m. Integrins exist in different activation states. When activating the integrin with Mg²⁺, we observe altered viscoelastic parameters of k_t = 0.9 μN/m, k_i = 190 μN/m, and μ = 6.0 μ N·s/m. Based on our model, we postulate that anchorage-related effects are common regulating mechanisms for cellular adhesion beyond affinity regulation.     

On Heels and Toes: How Ants Climb with Adhesive Pads and Tarsal Friction Hair Arrays

Ants are able to climb effortlessly on vertical and inverted smooth surfaces. When climbing, their feet touch the substrate not only with their pretarsal adhesive pads but also with dense arrays of fine hairs on the ventral side of the 3^rd and 4^th tarsal segments. To understand what role these different attachment structures play during locomotion, we analysed leg kinematics and recorded single-leg ground reaction forces in Weaver ants (Oecophylla smaragdina) climbing vertically on a smooth glass substrate. We found that the ants engaged different attachment structures depending on whether their feet were above or below their Centre of Mass (CoM). Legs above the CoM pulled and engaged the arolia (‘toes’), whereas legs below the CoM pushed with the 3^rd and 4^th tarsomeres (‘heels’) in surface contact. Legs above the CoM carried a significantly larger proportion of the body weight than legs below the CoM. Force measurements on individual ant tarsi showed that friction increased with normal load as a result of the bending and increasing side contact of the tarsal hairs. On a rough sandpaper substrate, the tarsal hairs generated higher friction forces in the pushing than in the pulling direction, whereas the reverse effect was found on the smooth substrate. When the tarsal hairs were pushed, buckling was observed for forces exceeding the shear forces found in climbing ants. Adhesion forces were small but not negligible, and higher on the smooth substrate. Our results indicate that the dense tarsal hair arrays produce friction forces when pressed against the substrate, and help the ants to push outwards during horizontal and vertical walking.     

Friction ridges in cockroach climbing pads: anisotropy of shear stress measured on transparent,microstructured substrates

The contact of adhesive structures to rough surfaces has been difficult to investigate as rough surfaces are usually irregular and opaque. Here we use transparent, microstructured surfaces to investigate the performance of tarsal euplantulae in cockroaches (Nauphoeta cinerea). These pads are mainly used for generating pushing forces away from the body. Despite this biological function, shear stress (force per unit area) measurements in immobilized pads showed no significant difference between pushing and pulling on smooth surfaces and on 1-μm high microstructured substrates, where pads made full contact. In contrast, on 4-μm high microstructured substrates, where pads made contact only to the top of the microstructures, shear stress was maximal during a push. This specific direction dependence is explained by the interlocking of the microstructures with nanometre-sized “friction ridges” on the euplantulae. Scanning electron microscopy and atomic force microscopy revealed that these ridges are anisotropic, with steep slopes facing distally and shallow slopes proximally. The absence of a significant direction dependence on smooth and 1-μm high microstructured surfaces suggests the effect of interlocking is masked by the stronger influence of adhesion on friction, which acts equally in both directions. Our findings show that cockroach euplantulae generate friction using both interlocking and adhesion.     

Egg adhesion of the codling moth Cydia pomonella L. (Lepidoptera, Tortricidae) to various substrates: I. Leaf surfaces of different apple cultivars

Codling moths, Cydia pomonella L. (Lepidoptera, Tortricidae), of the first generation deposit eggs on apple leaves in the vicinity of small fruits. The choice of the suitable oviposition sites and proper fixation of eggs are expected to be crucial factors for the survival of the offspring. In this study, we investigated egg adhesion of the codling moth to leaf surfaces of different cultivars of the domestic apple, Malus domestica Borkh., by measuring the pull-off force required to detach the eggs from leaves. Since surface features may influence insect egg adhesion, morphological and physicochemical properties (wettability, free surface energy) of these leaf surfaces were analyzed. Furthermore, eggs and their adhesives covering leaf surfaces were visualized. Eggs on the smooth upper leaf surfaces of all tested cultivars required significantly similar pull-off forces to be detached, at a total average of 6.0?mN. Up to 2?C3 times stronger pull-off forces had to be applied to detach eggs from trichome-covered lower leaves, and these forces differed significantly between cultivars. The role of leaf surface properties is discussed in the context of egg adhesion, oviposition site choice, female attachment, as well as neonate locomotion speed and survival. The obtained results shed light on the susceptibility of various apple cultivars and leaf surfaces to the infestation of apple trees by first-generation codling moths.     

Biomechanics of smooth adhesive pads in insects: influence of tarsal secretion on attachment performance

Many insects possess smooth adhesive pads on their legs, which adhere by thin films of a two-phasic secretion. To understand the function of such fluid-based adhesive systems, we simultaneously measured adhesion, friction and contact area in single pads of stick insects (Carausius morosus). Shear stress was largely independent of normal force and increased with velocity, seemingly consistent with the viscosity-effect of a continuous fluid film. However, measurements of the remaining force 2 min after a sliding movement show that adhesive pads can sustain considerable static friction. Repeated sliding movements and multiple consecutive pull-offs to deplete adhesive secretion showed that on a smooth surface, friction and adhesion strongly increased with decreasing amount of fluid. In contrast, pull-off forces significantly decreased on a rough substrate. Thus, the secretion does not generally increase attachment but does so only on rough substrates, where it helps to maximize contact area. When slides were repeated at one position so that secretion could accumulate, sliding shear stress decreased but static friction remained clearly present. This suggests that static friction which is biologically important to prevent sliding is based on non-Newtonian properties of the adhesive emulsion rather than on a direct contact between the cuticle and the substrate.     

Friction force reduction triggers feet grooming behaviour in beetles

In insects, cleaning (grooming) of tarsal attachment devices is essential for maintaining their adhesive ability, necessary for walking on a complex terrain of plant surfaces. How insects obtain information on the degree of contamination of their feet has remained, until recently, unclear. We carried out friction force measurements on walking beetles Gastrophysa viridula (Coleoptera, Chrysomelidae) and counted grooming occurrence on stiff polymer substrata with different degrees of nanoroughness (root mean square: 28-288 nm). Since nanoscopically, rough surfaces strongly reduced friction and adhesion without contaminating feet, we were able to demonstrate, for the first time to our knowledge, that friction force between tarsal attachment pads and the substrate provides an insect with information on the degree of contamination of its attachment structures. We have shown that foot grooming occurrence correlates not only with the degree of contamination but also with the decrease of friction force. This result indicates that insects obtain information about the degree of contamination, not statically but rather dynamically and, presumably, use mechanoreceptors monitoring either tensile/compressive forces in the cuticle or tensile forces between leg segments.     

Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow

L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. From velocity distributions of noninteracting and interacting microbeads, we observed that tether bond formation rates for P-selectin on PSGL-1 decreased with increasing wall shear stress, from 0.14 ± 0.04 bonds/μm at 0.2 dyn/cm² to 0.014 ± 0.003 bonds/μm at 1.0 dyn/cm². In contrast, L-selectin tether bond formation increased from 0.017 ± 0.005 bonds/μm at 0.2 dyn/cm² to 0.031 ± 0.005 bonds/μm at 1.0 dyn/cm². L-selectin tether bond formation rates appeared to be enhanced by convective transport, whereas P-selectin rates were inhibited. The transition force for the L-selectin catch-slip transition of 44 pN/bond agreed well with theoretical models (Pereverzev et al. 2005. Biophys. J. 89:1446-1454). Despite catch bond behavior, hydrodymanic shear thresholding was not detected with L-selectin beads rolling on PSGL-1. We speculate that shear flow generated compressive forces may enhance L-selectin bond formation relative to that of P-selectin and that L-selectin bonds with PSGL-1 may be tuned for the compressive forces characteristic of leukocyte-leukocyte collisions during secondary capture on the blood vessel wall. This is the first report, to our knowledge, comparing L-selectin and P-selectin bond formation frequencies in shear flow.     

Biological microtribology: anisotropy in frictional forces of orthopteran attachment pads reflects the ultrastructure of a highly deformable material

Evolutionarily optimized frictional devices of insects are usually adapted to attach to a variety of natural surfaces. Orthopteran attachment pads are composed of hexagonal outgrowths with smooth flexible surfaces. The pads are designed to balance the weight of the insect in different positions and on different materials. In a scanning electron microscopy study followed by freezing-substitution experiments, the ultrastructural architecture of the pad material was visualized. In friction experiments, the interaction was measured between the attachment pad and a polished silicon surface. The inner structure of this material contains distally directed rods, branching close to the surface, and spaces filled with fluid. The specific design of the pad material provides a higher frictional force in the distal direction. Frictional anisotropy is more enhanced at higher normal forces and lower sliding velocities. It is concluded that optimal mechanical functionality of biosystems is the result of a combination of surface structuring and material design.     

Toward physiological conditions for cell analyses: forces of heart muscle cells suspended between elastic micropillars

Almost each mammalian cell permanently applies forces to its environment. These forces are essential for many vital processes such as tissue formation or cell movement. In turn, the environmental conditions of cells strongly affect force production. Here we report on the development of an array of elastomeric micropillars as cellular environment. Within these micropillar arrays, we cultivated rat heart muscle cells (cardiac myocytes). For lattice constants between 20 and 30 μm, cells strongly preferred spanning between the elastic micropillars over adhering to the underlying flat substrate. In addition, the architectures of the cytoskeleton and of protein complexes formed for adhesion were strongly dependent on the environment of the cell. On flat parts of the substrates, we observed prominent stress fibers and focal adhesion sites. In contrast, cells suspended between micropillars exhibited well organized myofibers and costameric adhesions at the locations of Z-bands. These observations argue for close-to-nature environmental conditions within micropillar arrays. Resting as well as contraction forces of myocytes resulted in measurable pillar bending. Using an approximate theoretical treatment of elastically founded micropillars, we calculated average cell forces of 140 nN in the relaxed and 400 nN in the contracted state.     

Attachment ability of sawfly larvae to smooth surfaces

Larvae of the sawfly Rhadinoceraea micans adhere properly to the anti-adhesive surface of their host plant Iris pseudacorus by using three pairs of thoracic legs, seven pairs of abdominal prolegs, and pygopodia, all provided with various smooth adhesive pads. Their attachment performance to smooth flat hydrophilic and hydrophobic glass and Plexiglas surfaces was studied in centrifugal force experiments. Obtained safety factors on Plexiglas were up to 25 in friction, and 8 in adhesion. Although larvae attached significantly stronger to the hydrophilic glass, they attached well also to the hydrophobic one. Pygopodia are suggested to dominate attachment force generation in the centrifugal force experiment. Transverse body position on the centrifuge drum was significantly advantageous for friction force generation than was longitudinal body position. Results are discussed in the context of the sawfly biology and provide a profound base for further detailed studies on biomechanics of sawfly larvae–plant interactions.     

Insights into the mechanism of ADP action on flagellar motility derived from studies on bull sperm

Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 μM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 ± 0.29 nN/μm in 0.1 mM ATP and increasing to 2.9 ± 1.2 nN/μm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed.     

Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites

A frozen section method utilising chicken intestinal tissue was developed to study the Eimeria tenella attachment ex vivo. In order to examine Eimeria-epithelial cell attachment, 10⁵E. tenella sporozoites were incubated with each caecal frozen section (6, 10 and 14 μm) for 1 h in 5% CO₂ incubator at 41 °C. E. tenella sporozoites attached successfully to enterocytes in 14 μm thick of caecal sections. Sporozoite attachment to caecal sections was shown to be dependent on the number of parasites added. To evaluate the method, E. tenella sporozoites were incubated to its preferred (caecum) and non-preferred (duodenum and jejunum) intestinal sites. The number of sporozoites attached to the caecal enterocytes was significantly greater (P < 0.0001) in comparison with the limited number of sporozoites attached to enterocytes of non-preferred intestinal sites. This method was shown to be able to reveal differences in binding capability and allows for comparison of intestinal site attachment.     

A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth

Whether the axonal framework is stationary or moves is a central debate in cell biology. To better understand this problem, we developed a mathematical model that incorporates force generation at the growth cone, the viscoelastic properties of the axon, and adhesions between the axon and substrate. Using force-calibrated needles to apply and measure forces at the growth cone, we used docked mitochondria as markers to monitor movement of the axonal framework. We found coherent axonal transport that decreased away from the growth cone. Based on the velocity profiles of movement and the force applied at the growth cone, and by varying the attachment of the axonal shaft to the coverslip, we estimate values for the axial viscosity of the axon (3 × 10⁶ ± 2.4 × 10⁶ Pa·s) and the friction coefficient for laminin/polyornithine-based adhesions along the axon (9.6 × 10³ ± 7.5 × 10³ Pa·s). Our model suggests that whether axons elongate by tip growth or stretching depends on the level of force generation at the growth cone, the viscosity of the axon, and the level of adhesions along the axon.